Innovative PCR without DNA extraction for African sickle cell disease diagnosis

Objectives: Hemoglobin (Hb) disorders consist of thalassemia and Hb structural variants, of which the major forms are associated with severe anemia and/or vascular occlusion. Current diagnostic techniques are highly accurate and mostly based on isoelectric focusing, high-performance liquid chromatography or mass spectrometry, which often require advanced laboratory equipment. In sub-Saharan Africa, the Hb disorders are mainly associated to the pathological variants hemoglobin S (HbS) and HbC. Unfortunately, until now, it is not easy to get a diagnosis of these disorders in this area. In this study, we tested the performance of a new molecular diagnostic tests on qualified samples. Methods: The Human Hb S/C Lamp assay is a new polymerase chain reaction test able to detect HbS, HbC and HbA alleles without DNA extraction, directly on fresh or frozen blood samples, or on dried blood spots (DBS). In this study, we compared the genotyping of 248 blood samples (56 whole blood and 192 DBS) with this LAMP assay to the routine diagnostic methods performed in the genetics lab at the university hospital of Liege. Results: Our results show that the LAMP method can detect HbS and HbC with an accuracy of 100%. Moreover, this test can be used for the neonatal screening because we did not observe any interference with fetal Hb. Discussion: To our knowledge, this method is the only molecular assay that can be performed directly on dried blood cards without DNA extraction, lowering handling, turnaround time and costs.